Polo-like kinase 1 (Plk1) is a central component of the machinery that drives human cell division. Its function is required to prevent cells from gaining or losing chromosomes, which can lead to cancer. Conversely, it is also a promising target for cancer therapy. Plk1 executes multiple events in human cell division by binding and transferring a phosphate to substrate proteins. However, the precise phosphorylation events that are required remain obscure because they occur close together within the 60 minutes of human mitosis. Identification of these events can elucidate mechanisms of cytotoxicity, resistance, and unintended effects of Plk1-targeted drugs on normal cells. Innovation: Traditional genetic tools allow observation of the total effect of Plk1 loss on all substrates. In contrast, chemical genetics allows dissection of functional cross sections by abrogating phosphorylations of a subset of substrates. This approach has revealed new functions of Plk1 in division of human cells. The tools devised here will be useful for understanding functions of other multifunctional kinases. Approach: AIM 1 of this project seeks to elucidate Plk1 function in chromosome segregation. First, the functional significance of the Plk1 C-terminal polo-box domain in segregation will be tested by complementation assays. Next, the direct substrate responsible for missegregation will be identified. These findings will show how partial inhibition of Plk1 will affect dividing cells. AIM 2 will clarify Plk1-dependent events required to initiate cytokinesis. Initially this will focus on phosphorylation of Cyk4/RACGAP1, a component of the central spindle. The functional significance of these phosphorylations will be tested in biochemical assays and rescue experiments. In the second part of this AIM, the functional significance of additional Plk1 phosphorylations of other substrates will be tested for effect on cytokinesis. Outlook: The overall goal is to provide a comprehensive understanding of Plk1 function. This will provide crucial insight into the mechanisms of cytotoxicity for clinical Plk1 inhibitors, identify sensitive biomarkers of its inhibition, and pioneer new methodology to dissect multiple functions of other protein kinases-one of the most important classes of cancer drug targets.